For the last 40 years, almost all new protein structures have been solved by the multiple isomorphous replacement (MIR) method. To apply this method for the phase problem we need to prepare good heavy-atom derivatives, which is one of the most time-consuming process for the protein structure analysis, because it entails the trial-and-error experiments. Recently, an alternative approach has been devised for de novo phasing of protein crystals. This is the multiwavelengh anomalous diffraction(MAD). Especially important is the use of selenium atom for the anomalous scatterer, since it is easily incorporated into protein crystal by the protein engineering as a form of selenomethionine(Se-Met). In this project we applied this method to the analyses of several new proteins for further improvement of the method and showed that this method is very useful.Ribosomal protein 57 is located at the head of the small subunit facing to the decoding center. It is one of the primary 165 rRNA binding pro
… Moreteins responsible for initiating the assembly of the head of the 30S subunit. The structure of ribosomal protein S7 from Bacillus stearothermophilus has been solved at 2.5A resolution by the multiwavelength anomalous diffraction method using Se-Met substituted proteins. The molecule consists of a helical hydrophobic core domain and a beta-ribbon arm extending from the hydrophobic core. The helical core domain is composed of a pair of entangled helix-turn-helix motifs similar to a DNA architectural factor. Highly conserved basic and aromatic residues are clustered on one face of the S7 molecule and create a 165S rRNA contact surface. The molecular feature, together with former cross-link experiments, suggests how S7 binds to the 3' major domain of 16S rRNA and mediates the folding of 165 rRNA to create the ribosome decoding center.Ribosomal protein L2 is the largest protein component in the ribosome. It is located at or near the peptidyl transferase center and has been a prime candidate for the peptidyl transferase activity. It binds directly to 23 S rRNA and plays a crucial role in its assembly. The three dimensional structure of the RNA-binding domain of L2 from Bacillus stearothermophilus has been determined at 2.3A resolution by X-ray crystallography using the Se-Met MAD method. The RNA-binding domain of L2 consists of two recurring motifs of about 70 residues each. The residues Arg86 and Arg155, which have been identified by mutation experiments to be involved in the 23S rRNA binding, are located at the gate of the interface region between the two domains.The molecular architecture suggests how this important protein has evolved from the ancient nucleic acid binding proteins to create a 23S rRNA-binding domain in the very remote past. Less